Arthropod-transmitted flaviviruses are responsible for considerable morbidity and mortality, causing severe encephalitic, hemorrhagic, and febrile illnesses in humans. Because there are no specific clinical symptoms for infection by a determined virus and because different arboviruses could be present in the same area, a genus diagnosis by PCR would be a useful first-line diagnostic method. The six published Flavivirus genus primer pairs localized in the NS1, NS3, NS5, and 3 NC regions were evaluated in terms of specificity and sensitivity with flaviviruses (including the main viruses pathogenic for humans) at a titer of 10 5 50% tissue culture infectious doses (TCID 50 s) ml ؊1 with a common identification step by agarose gel electrophoresis. Only one NS5 primer pair allowed the detection of all tested flaviviruses with the sensitivity limit of 10 5 TCID 50 s ml ؊1 . Using a heminested PCR with new primers designed in the same region after an alignment of 30 different flaviviruses, the sensitivity of reverse transcription-PCR was improved and allowed the detection of about 200 infectious doses ml ؊1 with all of the tick-and mosquito-borne flaviviruses tested. It was confirmed that the sequenced amplified products in the NS5 region allowed predictability of flavivirus species by dendrogram, including the New York 99 West Nile strain. This technique was successfully performed with a cerebrospinal fluid sample from a patient hospitalized with West Nile virus encephalitis.

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Dengue virus type 2 and Yellow fever virus are arthropod-borne flaviviruses causing hemorrhagic fever in humans. Identification of virus receptors is important in understanding flavivirus pathogenesis. The aim of this work was to study the role of cellular heparan sulfate in the adsorption of infectious Yellow fever and Dengue type 2 viruses. Virus attachment was assessed by adsorbing virus to cells, washing unbound virus away, releasing cell-bound virus by freezing/thawing, and then titrating the released infectious virus. Treatment of cells by heparin-lyase, desulfation of cellular heparan sulfate, or treatment of the virus with heparin inhibited cell binding of both viruses. Heparin also inhibited Yellow fever virus infection by 97%. Using infectious virus, the present work shows the importance of heparan sulfate in binding and infection of these two flaviviruses.

The initial binding of Hepatitis C virus (HCV) to the cell membrane is a critical determinant of pathogenesis. Two putative HCV receptors have been identified, CD81 and low-density lipoprotein receptor (LDLr). CD81 interacts in vitro with the HCV E2 envelope glycoprotein, and LDLr interacts with HCV present in human plasma. In order to characterize these potential receptors for HCV, virus from plasma, able to replicate in cell culture, was inoculated on Vero cells or human hepatocarcinoma cells. HCV adsorption was assessed by quantitating cell-associated viral RNA by a real-time RT-PCR method. Anti-LDLr antibody, low and very low density lipoproteins inhibited significantly HCV adsorption, confirming the role of LDLr as HCV receptor. Only one out of the two anti-CD81 antibodies used in this study led to a partial inhibition of HCV binding. This study also highlights a role for glycosaminoglycans (GAGs) in HCV adsorption: treatment of virus with heparin led to 70% inhibition of attachment, as did desulfation of cellular GAGs. Treatment of Vero cells with heparin-lyase significantly inhibited virus attachment but by only 30%. These results demonstrate the complexity of the HCV binding step in which LDLr interacts strongly with HCV, whereas the interaction of HCV with GAGs and particularly with CD81 seem to be more moderate.