Grass carp reovirus (GCRV) is one of the most serious pathogens threatening grass carp (Ctenopharyngodon idella) production in China. Through sequence analysis, the co-existence of two genetically distant grass carp reoviruses, named GCRV-JX01 and GCRV-JX02, was revealed in the same diseased grass carp sample collected in 2011. GCRV-JX01 and GCRV-JX02 shared high levels of homology with GCRV-873 and GCRV-GD108, respectively. In contrast to GCRV-JX01, GCRV-JX02 induced no cytopathic effect in infected cells. A quantitative real-time PCR assay was employed to monitor the replication efficiency of both virus strains in either Ctenopharyngodon idella kidney (CIK) cells or infected cell supernatant. The results demonstrated that, although GCRV-JX02 did reduce the cellular replication level of GCRV-JX01 up to 10-fold during co-infection, there was no significant impact on the productive virus progeny level in supernatant compared to that of cells infected by GCRV-JX01 alone. To validate the hypothesis that both viruses might co-infect grass carp without significant interference in the field, we collected clinical samples from two different fish farms in 2012 and monitored virus loads for each fish. The data showed that 55 % of the collected fish samples were co-infected by GCRV-JX01 and GCRV-JX02, and the single virus infection rate was 10 % for GCRV-JX01 and 20 % for GCRV-JX02. For both viruses, the in vivo viral loads under co-infection and single viral infection were similar. No serological cross-reaction or cross-protection occurred between GCRV-JX01 and JX02 in our immunization and challenge tests. This new information on co-infection by two genetically distant virus strains should be helpful for designing vaccines targeting the causative agents of grass carp haemorrhagic disease. J Gen Virol. 2013 Jun94(Pt 6):1301-9. doi: 10.1099/vir.0.049965-0. Wang T, Li J, Lu L. Key Laboratory of Aquatic Genetic Resources of the Ministry of Agriculture, Shanghai Ocean University, 201306, PR China. (Provided by Fan Yuding).