B-70

M. Rios¹ , S. Daniel¹ , S. L. Stramer² , S. Caglioti³ , O. Wood¹ , I. K. Hewlett¹ , ¹LMV/DETTD/OBRR/CBER/FDA, Rockville, MD, ²American Red Cross, Gaithersburg, MD, ³Blood Systems Laboratory, Tampe, AZ

Blood units identified as WNV nucleic acid test (NAT) positive can be classified into 5 different stages of infection: I. early phase with very low viral load only detected sporadically positive by individual-NAT (ID-NAT) and IgM negative. II. Low viral load consistently detected by ID-NAT but negative for minipool-NAT (MP-NAT) and IgM; III. High viral load detectable by MP-NAT and IgM negative. IV. Decreasing viral load only detectable by ID-NAT and IgM positive; V. Convalescent phase with low viral load, negative by MP-NAT sporadically ID-NAT positive and IgM/IgG positive. Retrospective studies led to the identification of WNV transmission by transfusion. There are 30 cases of WNV transmission by transfusion, and retention samples from these donations all tested WNV ID-NAT positive and IgM negative. In addition, it was observed that most WNV low level RNA donations (MP-NAT negative and ID-NAT positive) also had IgM. That raises the question of whether donation ID-NAT and IgM positive but MP-NAT negative are infectious (transmits WNV to recipients). Currently there is no data on infectivity of such units. Animal models currently available do not have the capability of clarifying this issue. We investigated whether viremic plasmas containing IgM and/or IgG could infect susceptible cells in vitro. Vero cell and human monocyte-derived-macrophage (MDM) culture systems were used for infectivity studies of 48 WNV positive plasma samples to address this question. Cells were observed for cytopathic effect (CPE) on a daily basis. MDM cultures were used to test 19/48 plasmas and infectivity in MDM was assayed by RT-PCR-TaqMan for the WNV 3'NC region because WNV infection in MDM does not result in CPE. In the Vero cells infectivity assay 33/48 plasmas were CPE-positive by day 7, including 7 plasmas containing antibodies (3 IgM+ IgG-; 1 IgM- IgG+ and 3 IgM+ IgG+). RT-PCR-TaqMan results showed that 17/19 plasmas infected MDM cultures, including 4 plasmas that did not infect Vero cell

Serology Positive		Cell culture positive/total
		Vero cells	MDM	Combined
NAT	MP+ ID+	4/9	4/4	6/9
NAT	MP- ID+	2/7	2/4	4/7

In conclusion, WNV positive plasmas containing IgG and/or IgM are infectious for Vero cells and/or human MDM in vitro. Although, in vitro system does not represent in vivo situations the issue needs to be properly addressed. Therefore, non-human primate studies are needed to clarify the relevance of this finding in vivo.