Quantitative analysis of particles, genomes and infectious particles in supernatants of haemorrhagic fever virus cell cultures

Information on the replication of viral haemorrhagic fever viruses is not readily available and has never been analysed in a comparative approach. Here, we compared the cell culture growth characteristics of haemorrhagic fever viruses (HFV), of the Arenaviridae, Filoviridae, Bunyaviridae, and Flavivridae virus families by performing quantitative analysis of cell culture supernatants by (i) electron microscopy for the quantification of virus particles, (ii) quantitative real time PCR for the quantification of genomes, and (iii) determination of focus forming units by coating fluorescent antibodies to infected cell monolayers for the quantification of virus infectivity. The comparative analysis revealed that filovirus and RVFV replication results in a surplus of genomes but varying degrees of packaging efficiency and infectious particles. More efficient replication and packaging was observed for Lassa virus, and Dengue virus resulting in a better yield of infectious particles while, YFV turned out to be most efficient with only 4 particles inducing one FFU. For Crimean-Congo haemorrhagic fever virus (CCHFV) a surplus of empty shells was observed with only one in 24 particles equipped with a genome. The complete particles turned out to be extraordinarily infectious. Background Viral haemorrhagic fevers (VHF) are caused by various haemorrhagic fever viruses (HFV), of the Arenaviridae, Filoviridae, Bunyaviridae ,a ndFlavivridae virus families. Only few laboratories specialize in the research on these agents. Basic virological info rmation on these viruses is scant and described exclusively in the frame of case reports and pathological animal models. Although some progress has been achieved concerning the interaction of these viruses with mechanisms of innate immunity [1-9] and nitrite oxide pathways (CCHFV) [7] concise information on their basic virological characteristics is limited. This type of data however is important for biosafety risk assessment purposes. Here, we present a comparative analysis of quantities of HFV in cell culture determined by electron microscopic counting, quantitative real time RT-PCR and focus forming units, which reveal some features of the replication of these viruses that have not been described before. Methods Virus propagation