EVALUATION OF Murine norovirus (MNV)-1 INFECTION IN PRIMARY MOUSE B CELLS.

Abstract

Abstract Methods Primary mouse spleen B cells were stimulated or not with LPS (10ug/ml) + IL-4 (10IU/ml) for 48 hours. Two million cells were infected 72 h with MNV-1 at a MOI of 5. The non-structural (NS7) viral protein expression was analyzed in cell lysates by Western blot with a specific anti-NS7 antibody. The supernatant-associated virus in Raw 264.7 cells was determined by paque assay. Results B cells can be infected by MNV-1, even though they do not express the functional receptor CD300LD and CD300LF present in macrophages and Tuft cells. Since MNV-1 can uses CD44 as a receptor in BMDCs, we wanted to investigate if this molecule has a role in MNV-1 entry into B cells. First, we corroborated MNV-1 infection of B cells by evaluating the presence of NS7 by western blot and viral particles production in the supernatant at 72 hpi. To evaluate the role of CD44 in MNV-1 entry into B cells, we blocked the CD44 receptor using two different anti-CD44 monoclonal antibodies, and we found a significant decrease in both the amount of NS7 protein and virus yield, indicating that CD44 plays a role during virus binding/entry into B cells. Since CD44 expression is increased in LPS + IL-4 activated B cells, we wanted to determine if MNV-1 infection could be favored in these conditions. A correlation between the activation of B cells for 48 h and a significant increase of twice of MNV-1 NS7 protein and virus yield production were detected after infection. Conclusion Blocking CD44 correlates with a reduction of MNV-1 protein synthesis and virus yield production, indicating a role of CD44 in MNV-1 binding/entry. The increase of MNV NS7 protein and virus yield production in C57BL/6J spleen B cells activated with LPS + IL-4 that overexpresses CD44 on its surface, also supports this conclusion. CEMR is supported by a Conacyt fellowship, CVU fellowship number: 780860